Feline probiotic Lactobacilli

ABSTRACT

According to the invention there is provided a strain of lactic acid bacteria of the genus  Lactobacilli  obtainable by isolation from resected and washed feline gastrointestinal tract having a probiotic activity in animals. Methods of use and compositions comprising the  Lactobacilli  of the present invention are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/443,974, filed on May 31, 2006, which claims the benefit of andpriority to U.S. Provisional Application No. 60/692,440, filed on Jun.21, 2005, and U.S. Provisional Application No. 60/686,055, filed on May31, 2005.

FIELD OF THE INVENTION

The present invention relates to the field of probiotic micro-organisms,more specifically feline probiotic lactic acid bacteria and methods ofuse.

BACKGROUND OF THE INVENTION

The defense mechanisms to protect the mammalian gastrointestinal (GI)tract from colonisation by bacteria are highly complex. The GI tract ofmost mammals are colonised by native microflora, and invasive pathogenicmicro-organisms. In a healthy state, these competing microflora are in astate of equilibrium. Modification of the intestinal microfloraequilibrium may lead to or prevent many GI disorders, both in humans,and other mammalian species, such as companion animals including cats,dogs and rabbits. The well being of companion animals is closely relatedto their feeding and GI health, and maintenance of the intestinalmicroflora equilibrium in these animals may result in healthiercompanion animals.

The number and composition of the intestinal microflora tend to bestable, although age and diet may modify it. Gastric acidity, bile,intestinal peristalsis and local immunity are factors thought to beimportant in the regulation of bacterial flora in the small intestine ofhuman beings and various other mammals. Often companion animal GIdisorders, including those found in felines, are linked to bacterialovergrowth and the production of enterotoxins by pathogenic bacteria.These factors disrupt the intestinal microflora equilibrium and canpromote inflammation and aberrant immune responses.

During the last few years, research has begun to highlight some valuablestrains of bacteria and their potential use as probiotic agents.Probiotics are considered to be preparations of bacteria, either viableor dead, their constituents such as proteins or carbohydrates, orpurified fractions of bacterial ferments that promote mammalian healthby preserving the natural microflora in the GI tract, and reinforcingthe normal controls on aberrant immune responses. It is believed by somethat probiotic bacteria are more effective when derived from thespecies, or closely related species, intended to be treated. Therefore,there is a need for probiotic strains derived from companion animals tobe used for companion animals, that are different to those derived fromhumans.

WO 01/90311 discloses probiotic micro-organisms isolated from faecalsamples obtained from cats having probiotic activity. However, thesebacteria were obtained from faecal samples, and may not form part of thenatural intestinal microflora present in the upper portion of the GItract.

Consequently, there is a need to provide strains of bacteria obtainableby isolation from the natural intestinal microflora present in the upperportion of the GI tract that are particularly adapted for cats, and havebeen selected for their probiotic properties and ability to surviveprocessing, and to incorporate these strains into compositions that aresuitable for their use.

SUMMARY OF THE INVENTION

According to the invention there is provided strains of lactic acidbacteria of the genus Lactobacilli obtainable by isolation from resectedand washed feline gastrointestinal tract having a probiotic activity inanimals. The lactic acid bacterial strains are preferably selected fromthe species Lactobacillus salivarius, Lactobacillus animalis,Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillusjohnsonni Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacilluscasei, Lactobacillus cellobiosus, Lactobacillus curvatus, Lactobacillusdelbruekii, Lactobacillus fermentum, Lactobacillus helveticus,Lactobacillus lactis, or Lactobacillus plantarum.

In a preferred embodiment, the strain of lactic acid bacteria isselected from the group comprising Lactobacilli, having a 16s-23spolynucleotide sequence having greater than 94% homology to SEQ. ID NO.1, greater than 93% homology to SEQ. ID NO. 2, or greater than 98%homology to SEQ. ID NO. 3.

In a further preferred embodiment, the lactic acid bacterial strain isselected from the group comprising Lactobacillus salivarius sssalicinius NCIMB 41287 (AHF 122A), Lactobacillus animalis NCIMB 41288(AHF 223C), Lactobacillus reuteri NCIMB 41289 (AHF 5119) or a mutantthereof.

Furthermore, the present invention is directed towards providing uses oflactic acid bacteria obtainable by isolation from resected and washedfeline gastrointestinal tract for maintaining and improving companionanimal health, and compositions comprising the lactic acid bacteria.

These and other features, aspects, and advantages of the presentinvention will become evident to those skilled in the art from a readingof the present disclosure.

Sequences

SEQ. ID NO. 1—16s-23s intergenic spacer nucleotide sequence fromLactobacillus salivarius subsp. Salicinius, NCIMB 41287SEQ. ID NO. 2—16s-23s intergenic spacer nucleotide sequence fromLactobacillus animalis, NCIMB 41288SEQ. ID NO. 3-16s-23s intergenic spacer nucleotide sequence fromLactobacillus reuteri, NCIMB 41289SEQ. ID NO. 4—Left 16s-23s PCR primer sequence for sequence analysis.SEQ. ID NO. 5—Right 16s-23s PCR primer sequence for sequence analysis.

Bacterial Deposit Numbers

The table below indicates Lactobacillus species and strain number forstrains that are examples of the present invention. The bacterialstrains are deposited with the National Collections of Industrial Foodand Marine Bacteria (NCIMB), Aberdeen, UK.

16 s-23 s Strain Deposit Number Sequence Lactobacillus salivarius subsp.NCIMB 41287 SEQ. ID NO. 1 salicinius Lactobacillus animalis NCIMB 41288SEQ. ID NO. 2 Lactobacillus reuteri NCIMB 41289 SEQ. ID NO. 3

DETAILED DESCRIPTION OF THE INVENTION

All weights, measurements and concentrations herein are measured at 25°C. on the composition in its entirety, unless otherwise specified.

Unless otherwise indicated, all percentages of compositions referred toherein are weight percentages and all ratios are weight ratios.

Unless otherwise indicated, all molecular weights are weight averagemolecular weights.

Unless otherwise indicated, the content of all literature sourcesreferred to within this text are incorporated herein in full byreference.

Except where specific examples of actual measured values are presented,numerical values referred to herein should be considered to be qualifiedby the word “about”.

Within the following description, the abbreviation CFU (“colony-formingunit”) designates the number of bacterial cells revealed bymicrobiological counts on agar plates, as will be commonly understood inthe art.

As used herein, the term “mutants thereof” includes derived bacterialstrains comprising DNA mutations in other DNA sequences in the bacterialgenome excluding the 16s-23s intergenic sequence.

As used herein, the term “DNA mutations” includes natural or inducedmutations comprising at least single base alterations includingdeletions, insertions, transversions, and other DNA modifications knownto those skilled in the art, including genetic modification introducedinto a parent nucleotide or amino acid sequence whilst maintaining atleast 50% homology to the parent sequence. Preferably, the sequencecomprising the DNA mutation or mutations has at least 60%, morepreferably at least 75%, more preferably still 85% homology with theparental sequence. As used herein, sequence “homology” can be determinedusing standard techniques known to those skilled in the art. Forexample, homology may be determined using the on-line homology algorithm“BLAST” program, publicly available athttp://www.ncbi.nlm.nih.gov/BLAST/.

As used herein “genetic modification” includes the introduction ofexogenous and/or endogenous DNA sequences into the genome of an organismeither by insertion into the genome of said organism or by vectorsincluding plasmid DNA or bacteriophage as known by one skilled in theart, said DNA sequence being at least two deoxyribonucleic acid bases inlength.

As used herein, “companion animal” means a domestic animal. Preferably,“companion animal” means a domestic feline (cat), canine (dog), rabbit,ferret, horse, cow, or the like. More preferably, “companion animal”means a domestic feline.

Lactic Acid Lactobacilli Strains

The first aspect of the present invention comprises a strain of lacticacid bacteria of the genus Lactobacilli obtainable by isolation fromresected and washed feline gastrointestinal tract having probioticactivity in animals. Probiotics are micro-organisms, either viable ordead, processed compositions of micro-organisms, their constituents suchas proteins or carbohydrates, or purified fractions of bacterialferments that beneficially affect a host. The general use of probioticbacteria is in the form of viable cells. However, it can be extended tonon-viable cells such as killed cultures or compositions containingbeneficial factors expressed by the probiotic bacteria. This may includethermally killed micro-organisms, or micro-organisms killed by exposureto altered pH or subjected to pressure. For the purpose of the presentinvention, “probiotics” is further intended to include the metabolitesgenerated by the micro-organisms of the present invention duringfermentation, if they are not separately indicated. These metabolitesmay be released to the medium of fermentation, or they may be storedwithin the micro-organism. As used herein “probiotic” also includesbacteria, bacterial homogenates, bacterial proteins, bacterial extracts,bacterial ferment supernatants, and mixtures thereof, which performbeneficial functions to the host animal when given at a therapeuticdose.

It has been found that lactic acid bacteria of the genus Lactobacilliobtainable by isolation directly from resected and washed GI tract ofmammals are adherent to the GI tract following feeding of viablebacterial cells, and are also significantly immunomodulatory when fed toanimals in viable, non-viable or fractionated form. Without being boundby theory, it is believed that Lactobacilli obtainable by isolation fromresected and washed GI tract closely associate with the gut mucosaltissues. Without further being bound by theory, this is believed toresult in the probiotic Lactobacilli of the present invention generatingalternative host responses that result in its probiotic action. It hasbeen found that probiotic bacteria obtainable by isolation from resectedand washed GI tract can modulate the host's immune system via directinteraction with the mucosal epithelium, and the host's immune cells.This immunomodulation, in conjunction with the traditional mechanism ofaction associated with probiotic bacteria, i.e. the prevention ofpathogen adherence to the gut by occlusion and competition fornutrients, results in the Lactobacilli of the present invention beinghighly efficacious as a probiotic organism.

The Lactobacilli of the present invention, obtainable by isolation fromresected and washed feline GI tract, have in vitro anti-microbialactivity against a number of pathogenic bacterial strains/species.Without being bound by theory, it is believed that this in vitroanti-microbial activity is indicative of potential probiotic activity invivo in animals, preferably companion animals such as felines. Thelactic acid bacteria of the present invention preferably have in vitroanti-microbial activity against Salmonella typhimurium, Listeriamonocytogenes, Listeria innocua or Eschericia coli, more preferably amixture of these strains, more preferably still, all of these strains.

Without being bound by theory, it is believed that the anti-microbialactivity of the lactic acid bacteria of the present invention may be theresult of a number of different actions by the lactic acid bacteriaherein. It has previously been suggested in the art that several strainsof bacteria isolated from faecal samples exert their probiotic effect inthe GI tract following oral consumption by preventing the attachment ofpathogenic organisms to the gut mucosa by occlusion. This requires oralconsumption of “live” or viable bacterial cells in order for a colony ofbacteria to be established in the gut. However, it is believed that theLactobacilli of the present invention, obtainable by isolation fromresected and washed feline GI tract, whilst exerting some probioticeffect due to occlusion if given in a viable form, may deliver asubstantial probiotic effect in either the viable or non-viable form dueto the production during fermentation in vitro of a substance orsubstances that either inhibit the growth of or kill pathogenicmicro-organisms, and/or alter the host animal's immune competence. Thisform of probiotic activity is desirable, as the bacteria of the presentinvention can be given as either viable or non-viable cultures orpurified fermentation products and still deliver a beneficialtherapeutic effect to the host animal.

Preferably, the lactic acid bacteria of the present invention are ableto maintain viability following transit through the GI tract. This isdesirable in order for live cultures of the bacteria to be taken orally,and for colonisation to occur in the intestines and bowel followingtransit through the oesophagus and stomach. Colonisation of theintestine and bowel by the lactic acid bacteria of the present inventionis desirable for long-term probiotic benefits to be delivered to thehost. Oral dosing of non-viable cells or purified isolates thereofinduces temporary benefits, but as the bacteria are not viable, they arenot able to grow, and continuously deliver a probiotic effect in situ.As a result this may require the host to be dosed regularly in order tomaintain the health benefits. In contrast, viable cells that are able tosurvive gastric transit in the viable form, and subsequently colonise byadhering to and proliferating on the gut mucosa are able to deliverprobiotic effects continuously in situ.

Therefore, it is preferable that the lactic acid bacteria of the presentinvention maintain viability after suspension in a media having a pH of2.5 for 1 hour. As used herein, “maintain viability” means that at least25% of the bacteria initially suspended in the test media are viableusing the plate count method known to those skilled in the art.Preferably, “maintain viability” means that at least 50% of the bacteriainitially suspended are viable. It is desirable for the lactic acidbacteria of the present invention to maintain viability followingexposure to low pH as this mimics the exposure to gastric juices in thestomach and upper intestine in vivo following oral consumption inanimals.

Furthermore, it is preferable that the lactic acid bacteria of thepresent invention have a growth of at least 33% when in the presence ofat least 0.5% feline bile salts. Growth, as used herein is described infurther detail in example 3. More preferably, the bacteria of thepresent invention have a growth of at least 33% when in the presence ofat least 1% feline bile salts. More preferably still, the bacteria ofthe present invention have a growth of 100% in the presence of 0.5%feline bile salts. Without being bound by theory it is believed that thelactic acid bacteria of the present invention, capable of maintainingviability in the presence of at least 0.5% feline bile salts, are ableto survive the conditions present in the intestine. This is thought tobe a result of the addition of feline bile to the culture mediummimicking the conditions of the intestine.

Further still, it is preferable that the lactic acid bacteria of thepresent invention have significant adhesion to gut epithelial cells invitro. As used herein, “significant adhesion” means at least 1% of thetotal number of lactic acid bacteria co-incubated with the epithelialcells in vitro adhere to the epithelial cells. More preferably, at least5% of bacterial cells co-incubated adhere to epithelial cells in vitro.Without wishing to be bound by theory, it is believed that gutepithelial cell adherence in vitro is indicative of the lactic acidbacteria's ability to colonise the GI tract of an animal in vivo.

Preferably, the strain of lactic acid bacteria according to the presentinvention is of a species selected from the group comprisingLactobacillus salivarius, Lactobacillus animalis, Lactobacillus reuteri,Lactobacillus acidophilus, Lactobacillus johnsonni Lactobacillus brevis,Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluscellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii,Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis,or Lactobacillus plantarum. Preferably, the strain of lactic acidbacteria is selected from the group comprising Lactobacillus salivarius,Lactobacillus animalis, Lactobacillus reuteri, Lactobacillusacidophilus, or Lactobacillus johnsonni.

The 16s-23s intergenic polynucleotide sequence is known to those skilledin the art as the sequence of DNA in the bacterial genome that can beused in order to identify different species and strains of bacteria.This intergenic polynucleotide sequence can be determined by the methoddetailed below in example 4.

In a preferred embodiment of the present invention, the strain of lacticacid bacteria is selected from the group comprising Lactobacilli havinga 16s-23s polynucleotide sequence having greater than 94% homology toSEQ. ID NO. 1, greater than 93% homology to SEQ. ID NO. 2, or greaterthan 98% homology to SEQ. ID NO. 3. More preferably the strain of lacticacid bacteria is selected from the group comprising Lactobacilli havinga 16s-23s polynucleotide sequence having greater than 98% homology toSEQ. ID NO. 1, SEQ. ID NO. 2, or SEQ. ID NO. 3. More preferably still,the strain of lactic acid bacteria according to the present invention isselected from the group comprising Lactobacilli having a 16s-23spolynucleotide sequence selected from SEQ. ID NO. 1, SEQ. ID NO. 2, orSEQ. ID NO. 3. More preferably still, the strain of lactic acid bacteriaaccording to the present invention is selected from the group comprisingLactobacillus salivarius ss salicinius NCIMB 41287 (AHF 122A),Lactobacillus animalis NCIMB 41288 (AHF 223C), Lactobacillus reuteriNCIMB 41289 (AHF 5119) or a mutant thereof.

The strain of lactic acid bacteria of the genus Lactobacilli obtainableby isolation from resected and washed feline gastrointestinal tract canbe used to deliver probiotic benefit following oral consumption inanimals, preferably companion animals or humans. This probiotic benefitgenerally maintains and improves the overall health of the animal.Non-limiting elements of animal health and physiology that benefit,either in therapeutically relieving the symptoms of, or diseaseprevention by prophylaxis include inflammatory disorders,immunodeficiency, inflammatory bowel disease, irritable bowel syndrome,cancer (particularly those of the gastrointestinal and immune systems),diarrhoeal disease, antibiotic associated diarrhoea, appendicitis,autoimmune disorders, multiple sclerosis, Alzheimer's disease,amyloidosis, rheumatoid arthritis, arthritis, joint mobility, diabetesmellitus, insulin resistance, bacterial infections, viral infections,fungal infections, periodontal disease, urogenital disease, surgicalassociated trauma, surgical-induced metastatic disease, sepsis, weightloss, weight gain, excessive adipose tissue accumulation, anorexia,fever control, cachexia, wound healing, ulcers, gut barrier infection,allergy, asthma, respiratory disorders, circulatory disorders, coronaryheart disease, anaemia, disorders of the blood coagulation system, renaldisease, disorders of the central nervous system, hepatic disease,ischaemia, nutritional disorders, osteoporosis, endocrine disorders, andepidermal disorders. Preferred are treatment of the gastrointestinaltract, including treatment or prevention of diarrhoea; immune systemregulation, preferably the treatment or prevention of autoimmune diseaseand inflammation; maintaining or improving the health of the skin and/orcoat system, preferably treating or preventing atopic disease of theskin; ameliorating or reducing the effects of aging, including mentalawareness and activity levels; preventing disorders associated with thehypothalamus-pituitary-adrenal axis, and improving joint health wherebyimproving mobility.

The treatment of the disorders disclosed above may be measured usingtechniques known to those skilled in the art. For example, inflammatorydisorders including autoimmune disease and inflammation may be detectedand monitored using in vivo immune function tests such as lymphocyteblastogenesis, natural killer cell activity, antibody response tovaccines, delayed-type hypersensitivity, and mixtures thereof. Suchmethods are briefly described herein, but well known to those skilled inthe art.

-   -   1. Lymphocyte blastogenesis: This assay measures the        proliferative response in vitro of lymphocytes isolated from        fresh whole blood of test and control animals to various        mitogens and is a measure of overall T- and B-cell function.        Briefly, peripheral blood mononucleocytes (PBMC) are isolated        from whole blood by Ficoll-Hypaque density centrifugation        methods known to those skilled in the art. The isolated PBMCs        are washed twice in RPMI 1640 cell media supplemented with        HEPES, L-glutamine and penicillin/streptomycin. The washed cells        are resuspended in RPMI 1640, counted, and the cell density        adjusted appropriately. The 2×10⁵ cells are exposed to a range        of concentrations (0.1 μg/ml to 100 μg/ml) of various mitogens,        some examples of which include pokeweed mitogen (Gibco),        phytohaemagglutinin (Gibco) and conconavalin A (Sigma) in        triplicate for 72 hours at 37° C. and 5% CO₂ with 10% foetal        bovine serum (Sigma). At 54 hours the cells are pulsed with 1        μCi ³H-thymidine, and the cells harvested and scintillation        counts read on a TopCount NXT at 72 hours.    -   2. Natural killer cell activity: As described in U.S. Pat. No.        6,310,090, this assay measures the in vitro effector activity of        natural killer cells isolated from fresh whole blood of test and        control animals. Natural killer cells are a component of the        innate immune function of a mammal. Feline thyroid        adenocarcinoma cells were used as target cells in assessing NK        cell cytotoxic activity. This cell line was previously shown to        be susceptible to killing by feline NK cell. Target cells were        cultured in a T75 flask with 20 mL minimum essential medium        (MEM; Sigma Chem. Co., St. Louis, Mo.) supplemented with 10%        fetal calf serum (FCS), 100 U/mL of penicillin and 100 μg/mL of        streptomycin. When confluent, target cells were trypsinized,        washed 3 times and resuspended to 5×10⁵ cells/mL in complete        medium (RPMI-1640+10% FCS+100 U/mL of penicillin+100 μg/mL of        streptomycin). Triplicate 100.μL aliquots of the target cells        were pipetted into 96-well U-bottom plates (Costar, Cambridge,        Mass.) and incubated for 8 hours to allow cell adherence.        Lymphocytes (effector cells; 100.μL) isolated by Ficoll-Hypaque        separation (as described above) were then added to the target        cells to provide an effector/target cell (E:T) ratio of 10:1.        After 10 hours of incubation at 37° C., 20.μl of a substrate        containing 5.μg of        3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide        (MTT) was added. The mixture was incubated for 4 hours at 37° C.        after which the unmetabolized MTT was removed by aspiration. The        formazan crystals were dissolved by adding 200 μL of 95%        ethanol. Optical density was measured at 570 nm using a        microplate reader. The percentage of NK cell-specific lysis was        calculated as follows:

Specific Cytotoxicity (%)=100×{1−[(OD of target cells and effectorcells−OD of effector cells)/(OD of target cells)]}

-   -   3. Antibody response to vaccines: The test subjects are given an        array (up to 5) of vaccines after at least 12 weeks of probiotic        or control feeding. The vaccines may be a mixture of novel and        redundant vaccines. Non-limiting examples of vaccine arrays that        may be used include mixtures of vaccines prepared by Fort Dodge        Animal Health. Non-limiting examples of vaccines suitable for        use herein include Feline distemper, adenovirus, coronavirus,        parainfluenza, and parvovirus. The test subject's vaccine        history will determine the vaccines to be used. The specific        antibodies to the vaccines given are measured in blood for 3        weeks and the length and strength of response in control and        probiotic feeding groups compared.    -   4. Delayed-type hypersensitivity: An in vivo, non-invasive        method of assessing immune system status. This test comprises an        intradermal injection of the polyclonal mitogen        Phytohemmaglutinin (PHA) in combination with sheep red blood        cells a multivalent vaccine, histamine (100 μL of 0.0275 g/L        Histamine Phosphate; Greer, Lenoir, N.C.), or PBS (100 μL of        Phosphate Buffered Saline, 8.5 g/L; Sigma). The immune response        to the antigen is recorded as skinfold thickness using calipers        at time intervals of 0, 24, 48 and 72 hours post-injection. An        increase in skinfold thickness is indicative of a greater        hypersensitivity response that should be decreased by treatment        with the bacteria of the present invention.

Additional methods for determining the effect of the Lactobacillibacteria of the present invention are described in U.S. Pat. No.6,133,323 and U.S. Pat. No. 6,310,090.

Furthermore, ameliorating the effects of age may be determined usingdual x-ray absorptometry or CT scan for measuring body composition,including body fat mass, fat-free mass and bone mineral content.Similarly, this method may be used to determine anatomy changes such asweight loss or bone density in subjects following infection.

The Lactobacilli of the present invention may also be used in a methodfor reducing stress levels in companion animals. Concentrations of bloodstress hormones including epinephrine, norepinephrine, dopamine,Cortisol, C-reactive protein and other acute phase proteins may bemeasured to determine stress levels and their reduction or maintenance.These hormones are recognized biomarkers of stress and can be readilymeasured using techniques known to those skilled in the art.Additionally, direct measure of adrenal size as an in vivo marker ofactivity of the hypothalamus-pituatary-adrenal axis may be measured byCT imaging.

Further still, maintenance or improvement of the health of the skinand/or coat system of companion animals, including atopic disease of theskin, may be measured using skin and coat assessments conducted by twotrained individuals. Examples of criteria examined during suchassessments include:

-   -   a) Shedding index: A shedding index is assigned to each test        subject by collecting hair produced during a standardized        brushing session. The hair is retained and weighed, and control        and test subjects compared.    -   b) Subjective skin/coat evaluations: Trained panelists        subjectively evaluate skin and coat condition by assessing        shedding, dander, shine, uniformity, softness and density.    -   c) Skin functional assessment: The barrier function of the skin        may be assessed by wiping the skin surface with an        acetone-soaked gauze. This technique effectively disrupts the        skin barrier by removing single cell layers and associated lipid        fractions of the stratum corneum. Barrier disruption is        quantified by measuring the increase in transepidermal water        loss (TEWL) and the degree of redness of the insulted site using        methods known to those skilled in the art. Redness (erythema)        scores are obtained using the previously described camera and        lighting system. TEWL readings and redness scores are obtained        immediately before and after disruption, and at five and 24-hour        endpoints to assess the protective and healing properties of        skin.

The treatment or prevention of diarrhoea in companion animals may bemeasured using stool scores. Stools scores may be recorded dailyaccording to the following guidelines and control and test groupscompared before and after feeding with the bacteria according to thepresent invention.

Score: 5 Extremely Dry

This stool is hard and does not stick to surfaces. Stool will roll whenpushed. No indentations are made when stool is picked up. Stool is oftendefecated in groups of individual stools instead of one complete unit.The stool maintains original shape after collection.

Score: 4 Firm (Ideal Stool)

This stool is firm, well shaped, and cylindrical. This stool does notbreak apart easily when picked up. This stool may leave residue onsurfaces and gloves. This stool is often defecated as one unit. Thestool maintains original shape after collection.

Score: 3 Soft, with Shape

This stool is soft, however there are definite shapes. This stool willbreak apart easily and will definitely leave residue on surfaces andgloves. The stool often loses original shape after collection. Thisstool is often present with another score but can comprise whole stoolsample.

Score: 2 Soft, Without Shape

This stool is soft and will have no cylindrical shape. The shape oftenassociated with a “2” is a “cow patty” shape. This stool will lose theoriginal shape when collected and will definitely leave residue onsurfaces and gloves. This stool score is often present with anotherscore but can comprise the whole stool sample. This stool sample mayspread over an area of several inches.

Score: 1 Liquid

This stool score will always resemble liquid and there may or may not beparticulate matter present. This stool will often be defecated in groupsof piles instead of one complete unit. Mucous is often present with thisstool sample. This stool sample is very difficult to collect and residueis always left on surfaces and gloves. This stool sample may spread overan area of several inches.

In addition, other observations are also recorded, including: blood instool; foreign object in stool; or mucous in stool.

Furthermore, the treatment of gastrointestinal infection in companionanimals may comprise improving microbial ecology of companion animals.Improving the microbial ecology of companion animals preferablycomprises reducing the levels of pathogenic bacteria in the faeces ofcompanion animals. The levels of pathogenic bacteria present in thefaeces of companion animals may be enumerated using the standard platecount method known to those skilled in the art. More preferably, thepathogenic bacteria are selected from the group consisting ofClostridia, Escherichia, Salmonella, bacteriodes and mixtures thereof.Non-limiting examples of suitable strains of pathogenic bacteria includeC. perfringens, C. difficile, Eschericia coli, Salmonella typhimuriumand mixtures thereof.

The method of use of the bacteria of the present invention may alsoinclude the treatment, either prophylactic or therapeutic of the urinarytract of mammals, preferably companion animals. Non-limiting examples ofurinary tract treatment include treatment or prevention of urinary tractinfections, treatment or prevention of kidney disease, including urinarytract stones, treatment or prevention of bladder infections and thelike. Without being bound by theory, it is believed that theLactobacilli bacteria of the present invention are useful in preventingthese ailments as a result of their ability to degrade oxalic acid, asdemonstrated in vitro. Oxalic acid is a by-product of urinary metabolismthat can form insoluble precipitates that result in kidney, bladder andother urinary tract infections. By degrading oxalic acid, and thereforepotentially preventing its precipitation and build up in the urinarytract, the bacteria of the present invention may treat and preventinfections and other ailments of the urinary tract. Oxalic aciddegradation may be measured in vitro using the Oxalic acid test kit cat#755699 commercially available from Boehringer Mannheim/R-Biopharm.

The Lactobacilli of the present invention may be used in a method forimproving or maintaining the health of companion animals comprisingimproving fibre digestion. Improving fibre digestion is desirable as itpromotes the growth of said probiotic bacteria, as well as beneficialendogenous microflora, which aid in the suppression of some potentiallypathogenic bacteria. In addition, a decrease in the amount of toxicmetabolites and detrimental enzymes that result from colonicfermentation has been documented in humans (Tomomatsu, H. “Healtheffects of oligosaccharides”, (1994) Food Technol, 48, 61-65). Fibredigestion may be determined using the method described in Vickers et al.(2001), “Comparison of fermentation of selected fructooligosaccharidesand other fiber substrates by feline colonic microflora”, Am. J. Vet.Res. 61 (4), 609-615, with the exception that instead of inoculatingusing diluted fecal samples each experiment used pure cultures of thebacterial strains of interest.

The feline probiotic strains of the present invention may be used toreduce the odor of the feces and urine and concomitantly in thelitterbox by reducing the production of compounds in the feces and urinethat cause odor. Non-limiting examples of odor-causing compounds includeammonia, indoles, phenols, amines, branched chain fatty acids, andvolatile sulphur-containing compounds. Without wishing to be bound bytheory it is believed that reducing the levels o these compounds in thefeces or urine of a companion animal reduces the odor associated withthe feces or urine. Furthermore, for companion animals that use a litterbox, there is a concomitant decrease in litter box odor.

The method of use of the lactic acid bacteria of the present inventiontypically involves oral consumption by the animal. Oral consumption maytake place as part of the normal dietary intake, or as a supplementthereto. The oral consumption typically occurs at least once a month,preferably at least once a week, more preferably at least once per day.The lactic acid bacteria of the present invention may be given to thecompanion animal in a therapeutically effective amount to maintain orimprove the health of the animal, preferably a companion animal. As usedherein, the term “therapeutically effective amount” with reference tothe lactic acid bacteria, means that amount of the bacteria sufficientto provide the desired effect or benefit to a host animal in need oftreatment, yet low enough to avoid adverse effects such as toxicity,irritation, or allergic response, commensurate with a reasonablebenefit/risk ratio when used in the manner of the present invention. Thespecific “therapeutically effective amount” will vary with such factorsas the particular condition being treated, the physical condition of theuser, the duration of the treatment, the nature of concurrent therapy(if any), the specific dosage form to be used, the carrier employed, thesolubility of the dose form, and the particular dosing regimen.

Preferably, the lactic acid bacteria are given to the companion animalat a dose of from 1.0E+04 to 1.0E+14 CFU per day, more preferably from1.0E+06 to 1.0E+12 CFU per day. The composition preferably may containat least 0.001% of from 1.0E+04 to 1.0E+12 CFU/g of the lactic acidbacteria of the genus Lactobacilli obtainable by isolation from resectedand washed feline GI tract. The lactic acid bacteria can be given to theanimal in either viable form, or as killed cells, or distillates,isolates or other fractions of the fermentation products of the lacticacid bacteria of the present invention, or any mixture thereof.

Preferably, the lactic acid bacteria, or a purified or isolated fractionthereof, are used to prepare a composition intended to maintain orimprove the health of an animal. As indicated above, the composition maybe part of the normal dietary intake, or a supplement. Where thecomposition comprises part of the normal dietary intake, the compositionmay be in the form of a dried animal food such as biscuits or kibbles, aprocessed grain feed, a wet animal food, yoghurts, gravies, chews,treats and the like.

Such compositions may comprise further components. Other components arebeneficial for inclusion in the compositions used herein, but areoptional for purposes of the invention. For example, food compositionsare preferably nutritionally balanced. In one embodiment, the foodcompositions may comprise, on a dry matter basis, from about 20% toabout 50% crude protein, preferably from about 22% to about 40% crudeprotein, by weight of the food composition. The crude protein materialmay comprise any material having a protein content of at least about 15%by weight, non-limiting examples of which include vegetable proteinssuch as soybean, cotton seed, and peanut, animal proteins such ascasein, albumin, and meat tissue. Non-limiting examples of meat tissueuseful herein include fresh meat, and dried or rendered meals such asfish meal, poultry meal, meat meal, bone meal and the like. Other typesof suitable crude protein sources include wheat gluten or corn gluten,and proteins extracted from microbial sources such as yeast.

Furthermore, the food compositions may comprise, on a dry matter basis,from about 5% to about 35% fat, preferably from about 10% to about 30%fat, by weight of the food composition. Further still, food compositionscomprising the lactic acid bacteria of the present invention may alsocomprise from about 4% to about 25% total dietary fibre. Thecompositions may also comprise a multiple starch source as described inWO99/51108.

The compositions of the present invention may further comprise a sourceof carbohydrate. Grains or cereals such as rice, corn, milo, sorghum,barley, alfalfa, wheat, and the like are illustrative sources. Inaddition, the compositions may also contain other materials such asdried whey and other dairy by products.

The compositions comprising the bacteria of the present invention mayalso comprise a prebiotic. “Prebiotic” includes substances or compoundsthat are fermented by the intestinal flora of the companion animal andhence promote the growth or development of lactic acid bacteria in thegastro-intestinal tract of the companion animal at the expense ofpathogenic bacteria. The result of this fermentation is a release offatty acids, in particular short-chain fatty acids in the colon. Thishas the effect of reducing the pH value in the colon. Non-limitingexamples of suitable prebiotics include oligosaccharides, such as inulinand its hydrolysis products commonly known asfructooligosaccharides,galacto-oligosaccarides, xylo-oligosaccharides oroligo derivatives of starch. The prebiotics may be provided in anysuitable form. For example, the prebiotic may be provided in the form ofplant material which contains the fiber. Suitable plant materialsinclude asparagus, artichokes, onions, wheat or chicory, or residues ofthese plant materials. Alternatively, the prebiotic fiber may beprovided as an inulin extract, for example extracts from chicory aresuitable. Suitable inulin extracts may be obtained from Orafti SA ofTirlemont 3300, Belgium under the trade mark “Raftiline”. For example,the inulin may be provided in the form of Raftiline (g) ST which is afine white powder which contains about 90 to about 94% by weight ofinulin, up to about 4% by weight of glucose and fructose, and about 4 to9% by weight of sucrose. Alternatively, the fiber may be in the form ofa fructooligosaccharide such as obtained from Orafti SA of Tirlemont3300, Belgium under the trade mark “Raftilose”. For example, the inulinmay be provided in the form o Raftilose (g) P95. Otherwise, thefructooligosaccharides may be obtained by hydrolyzing inulin, byenzymatic methods, or by using micro-organisms.

For dried companion animal foods a suitable process is extrusioncooking, although baking and other suitable processes may be used. Whenextrusion cooked, the dried companion animal food is usually provided inthe form of a kibble. If a prebiotic is used, the prebiotic may beadmixed with the other ingredients of the dried companion animal foodprior to processing. A suitable process is described in European patentapplication No 0850569. If a probiotic micro-organism is used, theorganism is best coated onto or filled into the dried companion animalfood. A suitable process is described in European patent publicationNumber EP 0 862 863.

For wet foods, the processes described in U.S. Pat. Nos. 4,781,939 and5,132,137 may be used to produce simulated meat products. Otherprocedures for producing chunk type products may also be used; forexample cooking in a steam oven. Alternatively, loaf type products maybe produced by emulsifying a suitable meat material to produce a meatemulsion, adding a suitable gelling agent, and heating the meat emulsionprior to filling into cans or other containers. Typical wet foodcompositions may comprise from about 5% to about 15% protein, from about1% to about 10% fat, and from about 1% to about 7% fibre. Non-limitingingredients that may be used in wet food compositions include chicken,turkey, beef, whitefish, chicken broth, turkey broth, beef broth,chicken liver, brewers rice, corn grits, fish meal, egg, beet pulp,chloride, flax meal, lamb, beef by-products, chicken by-products andmixtures thereof.

In another embodiment, supplement compositions such as biscuits, chews,and other treats may comprise, on a dry matter basis, from about 20% toabout 60% protein, or from about 22% to about 40% protein, by weight ofthe supplement composition. As another example, the supplementcompositions may comprise, on a dry matter basis, from about 5% to about35% fat, or from about 10% to about 30% fat, by weight of the supplementcomposition. Food and supplement compositions intended for use byfelines or felines are commonly known in the art.

The companion animal foods may contain other active agents such as longchain fatty acids and zinc. Suitable long chain fatty acids includealpha-linoleic acid, gamma linolenic acid, linoleic acid,eicosapentanoic acid, and docosahexanoic acid. Fish oils are a suitablesource of eicosapentanoic acids and docosahexanoic acid.

Borage oil, blackcurrent seed oil and evening primrose oil are suitablesources of gamma linolenic acid. Safflower oils, sunflower oils, cornoils and soy bean oils are suitable sources of linoleic acid. These oilsmay also be used in the coating substrates referred to above. Zinc maybe provided in various suitable forms, for example as zinc sulfate orzinc oxide. Further, many ingredients commonly used in companion animalfoods are sources of fatty acids and zinc. It has been observed that thecombination of chicory, as a source of prebiotic, with a linoleic-acidrich oil, such as soy bean oil, provides unexpected benefits, suggestiveof a synergistic effect.

Where the composition is in the form of a gravy, the compositionpreferably comprises at least 10% of a broth, or stock, non-limitingexamples of which include vegetable beef, chicken or ham stock. Typicalgravy compositions may comprise from about 0.5% to about 5% crudeprotein, from about 2% to about 5% crude fat, and from about 1% to about5% fibre.

Further non-limiting examples of supplements suitable for use hereininclude powders, oil suspensions, milk-based suspensions, cheeses,cocoa-butter-based compositions and pills or capsules. Where thecomposition is in the form of a pill, suitable binding agents arerequired to maintain the pill in a solid, pressed form. Non-limitingexamples of suitable binding agents include the natural gums such asxanthan gum, pectins, lecithins, alginates and others known to thoseskilled in the art. Where the composition is in the form of a capsule,the composition is preferably encapsulated using technologies known tothose skilled in the art. Non-limiting examples of suitableencapsulation materials include polyvinyl alcohol (PVA),polyvinylpyrrolidone (PVP), alginates, and gelatin. Yoghurt-basedcompositions may comprise from about 1% to about 5% protein, from about10% to about 20% carbohydrate, from about 1% to about 5% fibre, fromabout 1% to about 5% fat and from about 50% to about 90% liquid carriersuch as milk.

EXAMPLES

The following examples are provided to illustrate the invention and arenot intended to limit the scope thereof in any manner.

Example 1 Isolation of Lactic Acid bacteria from feline GI tracts

Feline intestinal samples were obtained from healthy cats presenting atthe local veterinarians for owner initiated and approved euthanasia. Allanimals were healthy and disease-free. The colon, mid-colon, caecum andileum of each cat were dissected in order to expose the mucosa.

Supernatants were removed following agitation of the mucosal tissue(vortexed for 1 minute) and following mechanical homogenisation of thetissue. Each supernatant was plated on de Mann Rogosa Sharpe (MRS) agar.These were incubated anaerobically, using the Anerocult GasPak system,for 48 hours at 37° C. Isolated colonies from the plates werere-streaked onto either MRS and again grown anaerobically under the sameconditions. Isolated colonies were re-streaked a further 4 times inorder to purify a single strain. Colony morphology and microscopicappearance were assessed. Suitable isolates were tested for Gramreaction and catalase activity. Identification of gram positive,catalase negative rods was performed using API testing (API 50CHL,BioMerieux). Harvested cells were washed twice with 0.05M phosphatebuffer (pH 6.5) and cysteine-HCl (500 mg/i) followed by sonication.Centrifugation removed cellular debris. Supernatants were incubated withNaF (6 mg/ml) and Na iodoacetate (10 mg/ml) for 30 minutes at 37° C. Thereaction was stopped by incubation with hydroxylamine HCl (pH6.5) for 10minutes at room temperature. Colour development was monitored followingthe addition of HCl (4M), FeCl₃.6H₂O (5% (w/v) in 0.1M HCl) andfructose-6-phosphate (Na salt). Formation of acetyl phosphate fromfructose-6-phosphate was evidenced by the reddish colour formed by theferric chelate of its hydroxymate.

Example 2 Screening for Anti-Microbial Activity

Each of the isolated lactic acid bacterial strains was incubatedanaerobically in MRS broth. 2 μl of each culture were spotted onto MRSagar plates and incubated anaerobically overnight. Salmonellatyphimurium and Entero Pathogenic E. Coli (ExPEC) were pre-grownovernight and 100 μl inoculated into molten agar (1% v/v). Thisindicator culture was poured onto the surface of the inoculated MRSplates. Following overnight incubation, zones of inhibition around theprobiotic colony were measured. All experiments were performed induplicate on three separate occasions. In addition, incorporating thebuffer 2% betaglycerophosphate into the agar enabled assessment of thecontribution of acid production to the observed pathogen inhibition invitro.

The data presented in Table 2 clearly demonstrate that the lactic acidbacteria strains of the present invention obtainable by isolation fromresected and washed feline GI tract have significant anti-microbialactivity in vitro, indicative of potential probiotic activity.

TABLE 2 AHF122A AHF223C AHF5119 S. typhimurium 9.34 7.5 9.665 ExPEC11.84 7.67 11.17

Example 3 In Vitro Measures of Survival and Colonisation pH Tolerance

Bacterial cells were harvested from overnight cultures, washed twice inphosphate buffer (pH 6.5) and resuspended in MRS/TPY broth adjusted with1M HCl to pH 2.5. The cells were incubated anaerobically at 37° C. andtheir survival measured at intervals of 0, 30, 60, 120, 240 and 360minutes using the plate count method known to those skilled in the art.Table 3 summarises this data per strain.

TABLE 3 Survival of strains in a low pH environment (pH 2.5). All dataare log CFU counts. TIME (min) STRAIN 0 30 60 120 180 360 NCIMB 412879.31 9.13 8.95 8.88 8.84 8.52 NCIMB 41288 8.85 8.79 8.89 8.65 8.71 8.59NCIMB 41289 9.25 9.06 8.97 9.10 9.00 8.88

Bile Resistance

The bacterial strains were streaked onto MRS agar supplemented withporcine bile (Sigma) at 0.5%, 1% and 5% (w/v). Plates were incubated at37° C. under anaerobic conditions and the growth recorded after 48hours. Growth was compared with control plates by an experiencedobserver, and the growth of colonies described as:

Negative (0)—no growth;+(1)—Hazy translucent growth (<33% control-plates with 0% bile);++(2)—Definite growth but not as good as controls (>33% but <66%);+++(3)—Growth equivalent to controls (>66%).

Once the growth of the colonies in the presence of bile salts iscompared with the controls, the growth descriptors are given numericalvalues of 0, 1, 2 or 3 (−; +; ++, +++respectively), and then expressedas a percentage, where 3 represents 100%.

Table 4 demonstrates that the Bifidobacterium of the present inventionclearly demonstrate a resistance to bile salts, being able to grow andform colonies at a level of at least 66% in most instances when exposedto 0.3% porcine bile salts.

TABLE 4 Survival of strains in various concentrations of porcine bilePERCENTAGE BILE (%) STRAIN 0 0.3 0.5 1 2 5 7.5 10 NCIMB 41287 +++ + + −− − − − NCIMB 41288 +++ +++ +++ ++ ++ ++ ++ + NCIMB 41289 +++ +++ ++++++ ++ ++ + +

Furthermore, in order to assess any differences in the ability of thestrains to colonise the GI tract of cats, the bacterial strains werestreaked onto MRS agar supplemented with feline bile at 0.5%, 1% and 2%(w/v). Feline bile was obtained from cats undergoing endoscopy in aclinical setting during a non-terminal procedure. Plates were incubatedat 37° C. under anaerobic conditions and the growth recorded after 48hours. Growth was compared with control plates by an experiencedobserver, and the growth of colonies described as:

Negative (0)—no growth;

+(1)—Hazy translucent growth (<33% control-plates with 0% bile);

++(2)—Definite growth but not as good as controls (>33% but <66%);

+++(3)—Growth equivalent to controls (>66%).

Once the growth of the colonies in the presence of bile salts iscompared with the controls, the growth descriptors are given numericalvalues of 0, 1, 2 or 3 (−; +; ++, +++respectively), and then expressedas a percentage, where 3 represents 100%.

Table 5 demonstrates that the Bifidobacterium of the present inventionclearly demonstrate a resistance to feline bile salts, being able togrow and form colonies at a level of at least 66% in most instances whenexposed to 0.5% feline bile salts.

TABLE 5 Survival of strains in various concentrations of feline bilePERCENTAGE BILE (%) STRAIN 0 0.5 1 2 NCIMB 41287 +++ +++ +++ +++ NCIMB41288 +++ +++ ++ ++ NCIMB 41289 +++ +++ +++ +++

Gut Epithelial Cell Adhesion

The human epithelial cell line, HT-29, was used to assess the adhesionproperties of selected strains. Epithelial cells were routinely culturedas a monolayer in 75 cm² tissue culture flasks at 37oC in a humidifiedatmosphere containing 5% CO₂ in Dulbecco's Minimal Essential Media(DMEM) containing 10% foetal calf serum (FCS), pen/strep, glutamine andfungizone. For experimental purposes, the epithelial cells were seededat a concentration of 5×10⁵ cells/ml (3 mls total volume) per well in 6well culture plates (Sarstedt). Following incubation for 7 days, toallow differentiation, the epithelial monolayers were washed withantibiotic-free medium containing 10% FCS. Bacterial suspensions plus/inantibiotic-free DMEM were added to each well and the cells incubated for90 minutes at 37° C. Following incubation, the monolayers were washedthree times with PBS. The epithelial cells were lysed in deionised H2Oand the number of adherent bacteria enumerated using the plate countmethod known to those skilled in the art. Adhesion was expressed as apercentage of the number of bacteria initially plated. LactobacillusAHF122A had an adhesion level of 39.5%, whilst Lactobacillus AHF223C hadan adhesion level of 13.9%. Lactobacillus AHF5119 had an adhesion levelof 36.7%.

Example 4 16s-23s Intergenic Polynucleotide Sequencing

The feline Bifidobacterium isolates were centrifuged in stock tubes andthe resulting pellet lysed in 100 μl of Extraction Solution and 25 μl ofTissue Preparation solution (Sigma, XNAT2 Kit), incubated for 10 minutesat room temperature for 10 minutes. The samples were then incubated for5 minutes at 95° C. and then 100 □l of Neutralization Solution (XNAT2kit) was added. The genomic DNA solution was then, quantified using aNanodrop spectrophotometer and stored at 4° C.

PCR was performed using the intergenic spacer (IGS) primers, IGS L:5′-GCTGGATCACCTCCTTTC-3′ and IGS R: 5′-CTGGTGCCAAGGCATCCA-3′ Bridgidi etal 2000, System Appl. Microbiol., 23, 391-399 (2000)). The cyclingconditions were 94° C. for 3 min (1 cycle), 94° C. for 30 sec, 53° C.for 30 sec, 72° C. for 30 sec (28 cycles). The PCR reaction contained 4μl (50 ng) of DNA, PCR mix (XNAT2 kit), 0.4 μM IGS L and R primer (MWGBiotech, Germany). The PCR reactions were performed on an Eppendorfthermocycler. The PCR products (10 μl) were ran alongside a molecularweight marker (100 bp Ladder, Roche) on a 2% agarose EtBr stained gel inTAE, to determine the IGS profile.

PCR products of Bifidobacterium (single band) were purified using thePromega Wizard PCR purification kit.

The purified PCR products were sequenced using the primer sequences(above) for the intergenic spacer region. Sequence data was the searchedagainst the NCBI nucleotide database to determine the identity of thestrain by nucleotide homology.

Following sequencing, the obtained sequences for the four depositedstrains were compared with the on-line sequence database “BLAST”,available at http://www.ncbi.nlm.nih.gov/BLAST/ for homology with otherdeposited bacterial 16s-23s sequences. The closest match for AHF122A wasthe strain Lactobacillus salivarius subsp. Salicinius (AB102859) with ahomology score of 94%. The closest match for AHF223C was Lactobacillusanimalis strain LA51 (AY526615) with a homology score of 93%. Theclosest match for AHF5316 was Lactobacillus reuteri DSM 20016 (AF080100)with a homology score of 98%.

Example 5 Example Compositions

Examples 1 to 4 are examples of dried kibble compositions comprising theprobiotic Lactobacilli of the present invention.

Percentage on a weight Basis Ingredient Ex. 1 Ex. 2 Ex. 3 Ex. 4 Cerealgrains To 100 To 100 To 100 To 100 Poultry by-product meal 43.5 40 45 35Poultry fat 1.28 1.02 1.16 1.35 Egg product 2.4 2.1 2.5 2.2 Chickenliver meal 1.0 1.0 1.0 1.0 Brewer's dried yeast 1.0 1.0 1.0 1.0Monosodium phosphate 1.0 1.0 1.0 1.0 Calcium carbonate 0.8 0.8 0.8 0.8Potassium chloride 0.6 0.6 0.6 0.6 Vitamins 0.4 0.4 0.4 0.4 Cholinechloride 0.3 0.3 0.3 0.3 Minerals 0.3 0.3 0.3 0.3 DL-Methionine 0.1 0.10.1 0.1 Sodium Chloride 0.03 0.03 0.03 0.03 Probiotic (1 × 10¹⁰ cfu/g 10.5 — 0.6 NCIMB 41287 in sunflower oil) Probiotic (1 × 10¹⁰ cfu/g — 0.51 0.4 NCIMB 41288 in sunflower oil)

Examples 5 to 7 are examples of wet companion animal food compositionscomprising the probiotic Lactobacilli of the present invention.

Percentage on a weight Basis Ingredient Ex. 5 Ex. 6 Ex. 7 Water To 38 To47 To 50 Poultry Liver To 25 To 20 To 15 Poultry Products 25 20 20Brewers Rice 5 7 10 Egg Product 3 2.5 1.5 Poultry Fat 2.9 3.0 3.2Chicken Stock 0.6 0.7 0.9 Taurine 0.1 0.1 0.1 Vitamins 0.05 0.1 0.1Minerals 0.05 0.1 0.1 Probiotic (1 × 10¹⁰ cfu/g 4 5 6 NCIMB 41289)

Examples 8 to 10 are examples of yoghurt supplement compositionscomprising the probiotic Lactobacilli of the present invention.

Percentage on a weight Basis Ingredient Ex. 8 Ex. 9 Ex. 10 Milk 38 42 48Sugar 12 12 10 Modified Starch 1.0 0.8 0.8 Prebiotic 0.25 0.3 0.5Probiotic (1 × 10¹⁰ cfu/g 4 5 6 NCIMB 41287)

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1. A method of treating diarrhoea in a feline comprising orallyadministering to the feline a composition comprising a strain of lacticacid bacteria of the genus Lactobacilli in an amount effective to treatdiarrhoea in the feline.
 2. The method of claim 1 wherein the orallyadministering occurs as part of a normal dietary intake.
 3. The methodof claim 1 wherein the orally administering occurs as part of asupplement.
 4. The method of claim 1 wherein the orally administeringoccurs at least once per month.
 5. The method of claim 1 wherein thecomposition comprises a dose of from about 1E+04 CFU to about 1E+14 CFUper day of lactic acid bacteria.
 6. The method of claim 1 wherein thecomposition comprises at least 0.001% of from 1.0E+04 to 1.0E+12 CFU/gof the strain of lactic acid bacteria of the genus Lactobacilli.
 7. Themethod of claim 1 wherein the strain of lactic acid bacteria of thegenus Lactobacilli is in viable form or in the form of killed cells. 8.A method of preventing diarrhoea in a feline comprising orallyadministering to the feline a composition comprising a strain of lacticacid bacteria of the genus Lactobacilli in an amount effective toprevent diarrhoea in the feline.
 9. The method of claim 8 wherein theorally administering occurs as part of a normal dietary intake.
 10. Themethod of claim 8 wherein the orally administering occurs as part of asupplement.
 11. The method of claim 8 wherein the orally administeringoccurs at least once per month.
 12. The method of claim 8 wherein thecomposition comprises a dose of from about 1E+04 CFU to about 1E+14 CFUper day of the strain of lactic acid bacteria of the genus Lactobacilli.13. The method of claim 8 wherein the composition comprises at least0.001% of from 1.0E+04 to 1.0E+12 CFU/g of the strain of lactic acidbacteria of the genus Lactobacilli.
 14. The method of claim 8 whereinthe strain of lactic acid bacteria of the genus Lactobacilli is inviable form or in the form of killed cells.
 15. A method of increasingmobility in a feline comprising orally administering to the feline acomposition comprising a strain of lactic acid bacteria of the genusLactobacilli in an amount effective to increase mobility of the feline.16. The method of claim 15 wherein the orally administering occurs aspart of a normal dietary intake.
 17. The method of claim 15 wherein theorally administering occurs as part of a supplement.
 18. The method ofclaim 15 wherein the orally administering occurs at least once permonth.
 19. The method of claim 15 wherein the composition comprises adose of from about 1E+04 CFU to about 1E+14 CFU per day of the strain oflactic acid bacteria of the genus Lactobacilli.
 20. The method of claim15 wherein the composition comprises at least 0.001% of from 1.0E+04 to1.0E+12 CFU/g of the strain of lactic acid bacteria of the genusLactobacilli.
 21. The method of claim 15 wherein the strain of lacticacid bacteria of the genus Lactobacilli is in viable form or in the formof killed cells.
 22. A method of reducing the effects of aging in afeline comprising orally administering to the feline a compositioncomprising a strain of lactic acid bacteria of the genus Lactobacilli inan amount effective to reduce the effects of aging in a feline.